Membrane Environment Alters the Conformational Structure of the Recombinant Human Prion Protein

Morillas, Manuel; Świętnicki, Wiesław; Gambetti, Pierluigi; Surewicz, Witold K.

doi:10.1074/jbc.274.52.36859

Cited by 239 publications

(231 citation statements)

References 45 publications

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“…At pH 7.0, PrP displays almost no binding to POPC, while it binds in considerable amounts to POPC/Sm/GM1/Chol, with no particular preference for any of the three raft lipids tested. These results confirm those obtained by Morillas et al [24], monitoring tryptophan fluorescence, and using human prion protein and POPC liposomes, and also by Sanghera et al [25] using a fragment (90-231 residues) of PrP and dipalmitoylphosphatidylcholine/Chol/Sm liposomes. CD studies suggest that PrP at pH 7.0 maintains its native a-helical structure after incubation with liposomes, confirming results previously obtained with a different model system [25], and cross-linking experiments indicate that is almost completely monomeric.…”

Section: Discussionsupporting

confidence: 90%

Prion protein structure is affected by pH‐dependent interaction with membranes: A study in a model system

Sesana¹,

Barbiroli²,

Bonomi³

et al. 2007

FEBS Letters

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Interaction of full length recombinant hamster prion protein with liposomes mimicking lipid rafts or non-raft membrane regions was studied by circular dichroism, chemical cross-linking and sucrose gradient ultracentrifugation. At pH 7.0, the protein bound palmitoyloleoylphosphatidylcholine/ cholesterol/sphingomyelin/monosialoganglioside GM1 (GM1) ganglioside liposomes but not palmitoyloleoylphosphatidylcholine alone (bound/free = 0.33 and 0.01, respectively), maintaining the native a-helical structure and monomeric form. At pH 5.0, though still binding to quaternary mixtures, in particular GM1, the protein bound also to palmitoyloleoylphosphatidylcholine (bound/free 0.35) becoming unfolded and oligomeric. The pH-dependent interaction with raft or non-raft membranes might have implication in vivo, by stabilizing or destabilizing the protein.

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Section: Discussionsupporting

confidence: 90%

Prion protein structure is affected by pH‐dependent interaction with membranes: A study in a model system

Sesana¹,

Barbiroli²,

Bonomi³

et al. 2007

FEBS Letters

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“…In the presence of DOPG vesicles, there is a notable increase in negative ellipticity between 200 -235 nm and a shift in the minima of the spectrum from 208 to 205 nm. An increase in negative ellipticity in this region of the far-UV CD spectrum has been observed both for the human prion protein (27) and the ␦-endotoxin CytA (28) on binding phospholipid vesicles. In these cases, the observed changes in the far-UV CD spectrum were ascribed to a more ordered protein secondary structure.…”

Section: Negatively Charged Phospholipids Decrease the Stability Of Tmentioning

confidence: 96%

Destabilization of the Colicin E9 Endonuclease Domain by Interaction with Negatively Charged Phospholipids

Mosbahi

Walker

Lea

et al. 2004

Journal of Biological Chemistry

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We have shown previously that the 134-residue endonuclease domain of the bacterial cytotoxin colicin E9 (E9 DNase) forms channels in planar lipid bilayers (Mosbahi, K., Lemaître, C., Keeble, A. H., Mobasheri, H., Morel, B., James, R., Moore, G. R., Lea, E. J., and Kleanthous, C. (2002) Nat. Struct. Biol. 9, 476 -484). It was proposed that the E9 DNase mediates its own translocation across the cytoplasmic membrane and that the formation of ion channels is essential to this process. Here we describe changes to the structure and stability of the E9 DNase that accompany interaction of the protein with phospholipid vesicles. Formation of the protein-lipid complex at pH 7.5 resulted in a red-shift of the intrinsic protein fluorescence emission maximum ( max ) from 333 to 346 nm. At pH 4.0, where the E9 DNase lacks tertiary structure but retains secondary structure, DOPG induced a blue-shift in max , from 354 to 342 nm. Changes in max were specific for anionic phospholipid vesicles at both pHs, suggesting electrostatics play a role in this association. The effects of phospholipid were negated by Im9 binding, the high affinity, acidic, exosite inhibitor protein, but not by zinc, which binds at the active site. Fluorescence-quenching experiments further demonstrated that similar protein-phospholipid complexes are formed regardless of whether the E9 DNase is initially in its native conformation. Consistent with these observations, chemical and thermal denaturation data as well as proteolytic susceptibility experiments showed that association with negatively charged phospholipids destabilize the E9 DNase. We suggest that formation of a destabilizing protein-lipid complex preempts channel formation by the E9 DNase and constitutes the initial step in its translocation across the Escherichia coli inner membrane.Unraveling the interactions and mechanisms that enable proteins to cross biological membranes is of considerable interest, as the ability to target specific exogenous enzymes to the cytosol is likely to facilitate the design and discovery of novel chemotherapeutic agents. Many protein toxins have evolved to deliver a cytotoxic domain or subunit to the cytoplasm of susceptible cells, and so they provide an invaluable tool for studying protein translocation from the extracellular environment to their cellular targets, often located in the cytoplasm (1). The transition from the water-soluble to membrane-bound state has perhaps been most intensely studied in the pore-forming colicins (2, 3). This family of bacterial toxins, like all colicins, share a common three-domain structure, with receptor-binding and translocation domains that facilitate binding to the cell surface and mediate delivery of the channel-forming cytotoxic domain to the inner membrane. Cell death occurs as a consequence of ion channel formation across the cytoplasmic membrane, inducing depolarization of the membrane. Association of the cytotoxic domain with the membrane is thought to lead to destabilization and unfolding of the protein, yielding a "molten...

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“…The plasmid encoding huPrP90-231 with an N-terminal linker containing a His-6 tail and a thrombin cleavage site was described previously (45). The protein was expressed, cleaved with thrombin, and purified according to the previously described protocol (45); it was stored frozen in 10 mM sodium acetate buffer (pH 4).…”

Section: Methodsmentioning

confidence: 99%

β-Sheet core of human prion protein amyloid fibrils as determined by hydrogen/deuterium exchange

Wintrode²,

Surewicz

2007

Proc. Natl. Acad. Sci. U.S.A.

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212

261

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Propagation of transmissible spongiform encephalopathies is associated with the conversion of normal prion protein, PrP C , into a misfolded, oligomeric form, PrP Sc . Although the high-resolution structure of the PrP C is well characterized, the structural properties of PrP Sc remain elusive. Here we used MS analysis of H/D backbone amide exchange to examine the structure of amyloid fibrils formed by the recombinant human PrP corresponding to residues 90 -231 (PrP90 -231), a misfolded form recently reported to be infectious in transgenic mice overexpressing PrP C . Analysis of H/D exchange data allowed us to map the systematically H-bonded ␤-sheet core of PrP amyloid to the C-terminal region (staring at residue Ϸ169) that in the native structure of PrP monomer corresponds to ␣-helix 2, a major part of ␣-helix 3, and the loop between these two helices. No extensive hydrogen bonding (as indicated by the lack of significant protection of amide hydrogens) was detected in the N-terminal part of PrP90 -231 fibrils, arguing against the involvement of residues within this region in stable ␤-structure. These data provide long-sought experimentally derived constraints for high-resolution structural models of PrP amyloid fibrils.prion diseases ͉ transmissible spongiform encephalopathy ͉ amyloid structure ͉ mass spectrometry

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Membrane Environment Alters the Conformational Structure of the Recombinant Human Prion Protein

Cited by 239 publications

References 45 publications

Prion protein structure is affected by pH‐dependent interaction with membranes: A study in a model system

Prion protein structure is affected by pH‐dependent interaction with membranes: A study in a model system

Destabilization of the Colicin E9 Endonuclease Domain by Interaction with Negatively Charged Phospholipids

β-Sheet core of human prion protein amyloid fibrils as determined by hydrogen/deuterium exchange

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